Schenk, Gerhard and Leeper, Finian J. and England, Renee and Nixon, Peter and Duggleby, Ronald
The role of His113 and His114 in pyruvate decarboxylase
from Zymomonas mobilis.
European Journal of Biochemistry , 248.
Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP)
and a divalent cation as essential cofactors. Recently, the three-dimensional structures of the enzyme from
two yeasts have been determined. While these structures shed light on the binding of the cofactors and
the reaction mechanism, the interactions between the substrate pyruvate and the enzyme remain unclear.
We have used PDC from Zynzomonas mobilis as a model for these enzymes in order to study substrate
binding. The recombinant enzyme was expressed in Escherichia coli. High yield, simplicity of purification,
high stability and simple kinetics make this model well suited for these studies. Activity measurements
in the pH range between 5.8 and 8.5 indicated that a His residue may be involved in substrate
binding. Analysis of an alignment of all known PDC protein sequences showed two invariant His residues
(His113 and Hisll4) which, according to the crystal structure of yeast PDC, are in the vicinity of the
active site. Here we demonstrate that replacement of His114 by Gln does not have a great effect on
cofactor and substrate binding. However, the k,,, is decreased indicating that Hisl 14 may assist in catalysis.
In contrast, substitution of His113 by Gln renders the enzyme completely inactive. This mutant has
decreased affinity for both cofactors, as revealed by measurements of tryptophan fluorescence quenching.
However, this decreased affinity is insufficient to account for the complete loss of activity. Despite its
inability to support overall catalysis, this [Glnl13]PDC mutant is capable of releasing acetaldehyde from
2-( 1 -hydroxyethyl)thiamin diphosphate supplied exogenously. It is proposed that upon substrate binding,
His113 is placed close to C2 of the thiazole ring. Subsequent deprotonation of this atom leads to a
conformational change that allows a flexible loop (residues 105-112) that precedes Hisl 13 to close over
the active site. Hence, replacement of His113 by another residue interferes with this closure of the active
site and thus disrupts the catalytic process.
||pyruvate decarboxylase; Zyrnornonas mobilis; site-directed mutagenesis; substrate binding; catalysis;
||Faculty of Science and Engineering > Chemistry
||30 May 2012 09:04
|Journal or Publication Title:
||European Journal of Biochemistry
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